Chemokines are small chemotactic cytokines, which direct cellular migration through receptor specific interactions on target cells (Arya, M., et al. (2003). Curr. Med. Res. Opin. 19, 557-564). The homeostatic chemokine-chemokine receptor pair CXCL12 and CXCR4 is widely expressed throughout the body (Bleul, C. C., et al. (1996) J. Exp. Med. 184, 1101-1109). CXCL12, formerly known as stromal cell-derived factor-1 (SDF1), is an alpha type 7.8 kDa CXC chemokine (Shirozu, M., et al. (1995) Genomics 28, 495-500). Originally described as a growth factor for bone marrow developing B cells (Nagasawa, T., et al. (1994) Proc. Natl. Acad. Sci. U.S.A 91, 2305-2309), CXCL12 was subsequently characterized as a chemoattractant for T cells and monocytes (Bleul, C. C., et al. (1996) J. Exp. Med. 184, 1101-1109). Genetic ablation of CXCR4 or CXCL12 results in embryonic lethality (Nagasawa, T., et al. (1996) Nature 382, 635-638). Similar embryonic defects in either of those chemokine receptor or chemokine gene deficient animals has revealed roles for CXCR4-CXCL12 signaling in cardiovascular, neuronal, and hematopoietic stem cell development as well as gastrointestinal vascularization (Tachibana, K., et al. (1998) Nature 393, 591-594; Zou, Y. R., et al. (1998) Nature 393, 595-599). Previous studies by our group have established a role for CXCL12 and CXCR4 in gut vascularization, a key process in mucosal immunity and homeostasis (Heidemann, J., et al. (2004) Am. J. Physiol Gastrointest. Liver Physiol 286, G1059-G1068).
In addition to endothelial expression, the cells of the human colonic epithelium also express both CXCL12 and CXCR4 (Jordan, N. J., et al. (1999) J. Clin. Invest 104, 1061-1069; Agace, W. W., et al. (2000) Curr. Biol. 10, 325-328; Dwinell, M. B., et al. (1999) Gastroenterology 117, 359-367.). Moreover, using an in vitro wound healing assay we have shown that non-transformed intestinal epithelial cells migrate across a denuded surface in response to CXCL12, a key component of the rapid healing ability of the mucosal epithelial surface (Smith, J. M., et al. (2005) Am. J. Physiol Gastrointest. Liver Physiol 288, 316-26). The role of CXCL12-CXCR4 signaling in mucosal wound healing is consistent with other physiologic processes utilizing this signaling axis such as organogenesis and immune surveillance. Thus, our data demonstrate an important role for the combined expression of both CXCR4 and CXCL12 by the cells of the mucosal epithelium. More broadly, these processes of epithelial wound healing, enterocyte migration and vascular angiogenesis, which we have shown in healthy gut mucosa, are known to be dysregulated in colorectal cancer as well as chronic inflammatory diseases. Recent evidence indicates that CXCR4 expression by carcinoma cells may also participate in the metastasis of various cancer types including breast, prostate, non-small cell lung, and colon (Muller, A., et al. (2001) Nature 410, 50-56; Sun, Y. X., et al. (2003) J. Cell Biochem. 89, 462-473, 2003; Phillips, R. J., et al. (2003) Am. J. Respir. Crit. Care Med. 167, 1676-1686; Zeelenberg, I. S., et al. (2003) Cancer Res. 63, 3833-3839). Notably, several studies linking chemokine receptor signaling to cancer cell metastasis suggest that aberrant regulation of CXCR4 expression plays an important role in this process (Haviv, Y. S., et al. (2004) Mol. Cancer. Ther. 3, 687-691; Muller, A., et al. (2001). Nature 410, 50-56.). In contrast, studies defining CXCL12 expression in various carcinomas are more limited.
Given the important functional roles and consistent dual expression of both CXCR4 and CXCL12 by human intestinal epithelium (Dwinell, M. B., et al. (1999). Gastroenterology 117, 359-367; Agace, W. W., et al. (2000) Curr. Biol. 10, 325-328) we hypothesized that perturbations in epithelial CXCL12 expression would contribute to colorectal carcinoma disease progression, possibly by allowing carcinoma cells to more readily sense CXCL12 from exogenous sources, aiding metastasis. This novel hypothesis is supported by evidence from developing hematopoietic stem cells exiting the bone marrow, in which a disruption of CXCL12-CXCR4 signaling is required for stem cell mobilization (Christopherson, K. W., et al. (2004) Science 305, 1000-1003; Gazitt, Y. (2004) Leukemia 18, 1-10). Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies. Leukemia 18, 1-10).
Our data herein suggest that a disruption in CXCR4 autocrine signaling results from the silencing of CXCL12 in human colonic carcinoma cells. We define a mechanism of CXCL12 silencing in human colorectal carcinoma by DNA methyltransferase (Dnmt) enzyme mediated promoter hypermethylation. Consistent with our hypothesis, re-establishment of endogenous CXCL12 expression in colonic carcinoma cells dramatically reduced in vivo metastatic tumor formation. Our data demonstrate a previously unrecognized mechanism of CXCL12 silencing in colorectal carcinoma, which significantly impacted the metastatic properties of those cells. Further, we suggest a new paradigm in which the epigenetic silencing of one arm of the CXCL12-CXCR4 signaling axis promotes tumor cell metastasis.